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The authors declare that the data supporting the findings of 1684 study are available within the article and its Supplementary Information files, or are available from the authors upon request.
Current influenza vaccines provide limited protection against circulating influenza A viruses. A universal influenza vaccine will eliminate the intrinsic limitations of the seasonal flu vaccines. Here we report methodology to generate double-layered protein nanoparticles as a universal influenza vaccine.
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Layered nanoparticles are fabricated by desolvating tetrameric M2e into protein nanoparticle cores and 11684 these cores by crosslinking headless HAs. Representative headless HAs of two HA phylogenetic lwi are constructed and purified. Vaccinations with the resulting protein nanoparticles in mice induces robust long-lasting immunity, fully protecting the mice against challenges by divergent influenza A viruses of the same group or both groups.
The results demonstrate the importance of incorporating both structure-stabilized HA stalk domains and M2e into a universal influenza vaccine to improve its protective potency and breadth.
These potent disassemblable protein nanoparticles indicate a wide application in protein drug delivery and controlled release. Mutant viruses acquire the ability to escape from leei herd immunity by antigenic drift and shift, which necessitates the yearly update of the composition of seasonal influenza vaccines to match the newly circulating viruses 1.
The protective efficacy of the seasonal vaccines does not always live up to expectation. The outbreak of H1N1 pandemic causeddeaths during the first 12 months of its leei 2. Low vaccine effectiveness was also observed recently during the — and — flu seasons 34.
The sporadic human cases of fatal zoonotic H5N1 and H7N9 infections are also serious public health threats 5 — 7. A universal influenza vaccine which induces broad cross protection against divergent viruses is urgently needed to eliminate these threats.
Conserved determinants from influenza antigenic proteins are potential immunogens for such universal influenza vaccines.
The HA stalk domain is relatively conserved compared to the variable globular head domain 89. Accompanying the isolation and artificial generation of broadly neutralizing antibodies 10 — 16some HA stalk domain-based immunogens have been constructed and proven protective to some extent in vivo 17 — The amino acid sequence of influenza matrix protein 2 ectodomain M2e is highly conserved among human seasonal influenza A viruses Natural human influenza A virus infections induce only weak anti-M2e antibody responses of short duration A possible explanation for this low immunogenicity is the small size of M2e and the low abundance of M2 in virions compared to the large glycoproteins, HA, and NA Therefore, M2e is often constructed with a larger carrier or presented as a soluble tetramer antigen to enhance anti-M2e immune responses in vaccination experiments 23 Multiple copies of M2e in a construct can dramatically enhance the M2e specific antibody responses Clinical trials have demonstrated that M2e based vaccines are safe and immunogenic in humans 2026 Human passive immunization with humanized anti-M2e monoclonal antibody TCN proved to be efficient in reducing virus replication, demonstrating the effectiveness of vaccine-driven anti-M2e antibody-based immunity Clinical trial results have shown that the overall induced M2e antibody responses in M2e-HBc vaccinated volunteers faded away rapidly within 10 months Successful applications of nanotechnology hold great promise for the development of new generations of influenza vaccines.
Large self-assembling motifs can enable mer 1729 and even mer 30 protein nanoparticle PNp assembly. However, self-assembly motifs increase the risk of off-target immune responses due to their high immunogenicity. Desolvated PNp core coated with viral antigen on the surface represents a convenient solution to these issues and does not require encapsulation materials. In this study, we found that layered PNps composed of structure-stabilized HA stalk domains from both HA groups, and novel constructed M2e, are highly immunogenic to induce immune protection against homosubtypic and heterosubtypic influenza A virus challenges.
The double-layered PNps have the potentials to be developed into a universal influenza vaccine. The physiologically activated disassembly of PNps after the uptake into cells implies a wide utilization for protein drug delivery and controlled release. We successfully constructed and expressed the structure-stabilized soluble tetramer protein containing four tandem copies of M2e 4MtG and trimeric head-removed hr HAs from representatives of both HA phylogenetic groups designed hrH1 and hrH3.
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To increase the potency and breadth of protection, 4MtG includes four types of M2e from human, swine, avian, and domestic fowl viral consensus sequences Fig. The generation of hrHA representatives from both phylogenetic groups is essential to formulate a vaccine that can elicit a genuinely universal protective response. Recombinant protein construction and PNp generation and characterization. Blue arrows indicate the location of flexible linkers.
Dashed lines indicate the sequences replaced with linkers shown below. Brown arrows indicate the site-mutations, VC and SC. Due to its metastable conformation, HA2 expressed independently from the HA1 subunit will spontaneously adopt a low-pH conformation This indicates that the recombinant hrH1 and hrH3 proteins were folded in a similar fashion to the corresponding region in HA in the neutral pH structure.
This approach produced PNps composed almost entirely of the antigens of interest and did not include carriers, avoiding off-target immune responses to vectors 1729 To minimize the PNp size, the core was not crosslinked until after the addition of soluble hrHA during the coating process because proteins self-assembled into smaller PNps in the absence of a crosslinker Supplementary Fig.
Scanning electron micrograph SEM showed that the particles were relatively spherical with irregular surface morphology Fig. There was no significant difference among the three nanoparticle types Supplementary Fig. Intensity analysis of the protein bands in Coomassie Blue stained gel estimated the overall percentages of hrH1 and hrH3 coatings to be The unbound hrHA molecules and self-crosslinked hrHAs cannot be pelleted by high-speed centrifugation To evaluate the protective efficacy of the resulting PNp vaccines, mice were intramuscularly immunized twice with PNps in the absence of adjuvants.
IgG2a-biased antibody responses were induced, as evidenced by the IgG1: IgG2a ratio Supplementary Fig.
Humoral and cellular immune responses of vaccinated mice. Because frequent human infection with fatal zoonotic influenza H5N1, H7N9, and H10N8 viruses in recent years represents possible emerging pandemics, the binding activities of immune sera to H5, H7, and H10 were tested.
The waning human herd immunity against H2N2 influenza strain and low mutation rate make it likely that a new pandemic could arise from the current circulating H2N2 strain among birds and swine. H2 was included in the serum binding assay.
The squares besides HA subtypes indicate HA phylogenetic groups by coloring: P values shown in bar charts and N.
The experiments were repeated lek with similar results. Cellular responses are also important for the generation and 111684 of effective immunity and contribute to the killing of pathogens 37 As shown in Fig. Prophylaxis potency lel evaluated by challenge studies. Uni4C1 and Uni4C3 immunizations conferred complete homologous protection against death and weight loss Fig. The conservation between divergent subtypes of HA is quite limited Fig.
Moreover, the Uni4C13 immunization dramatically reduced lung virus titers at day 5 post infection compared to the mock-immunization Fig. Histological analysis showed significantly decreased leukocyte infiltration in infected lungs from Uni4C13 immunized mice Fig.
PNp protective efficacy in mice. Leu body weight changes and survivals upon lethal dose challenges. Mouse body weight changes and survivals were monitored for 2 weeks. Amino acid differences in HA lel. The HA1 domain is shown in green-cyan and the HA2 domain is shown in blue. Lung physiology in virus challenge infection. Uninfected lung section is used as negative control. Images from c and d are representatives from five individual mouse lung sections each group.
We next investigated the antibody-mediated effector mechanisms. NT activity against rSH was undetectable. oei
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Virus neutralization potency of immune sera was evaluated in a standard neutralization assay against the same panel of influenza viruses as in the HAI assays.
Besides direct viral NT, Fc-mediated effector mechanisms such as antibody dependent cellular cytotoxicity ADCC contribute substantially to protection against influenza challenges 10 In contrast to mice that received mock-sera, mice that received sera from Uni4Cimmunized mice were protected significantly, indicating that serum antibodies played critical roles in the protection conferred by Uni4C Correlates of immune Protection.
The non-transfected HEKT cells were used as negative control. Other two bars show the anti-M2e IgG endpoint titers in PBS loaded or clodronate-loaded liposomes treated recipient mice.
Body weight loss and survival rate were monitored for 14 days. Statistical significance analysis of survival rates was performed by Kaplan-Meier analysis for d — g.
Data are representative from two independent experiments. Clodronate-loaded liposomes were intra-tracheally administered for selective depletion of alveolar macrophages AM. Challenge results showed that only mice receiving Uni4C13 serum and without the AM depletion were fully protected against lethal dose Phi H3N2 challenge, indicating an antibody-dependent cellular phagocytosis ADCP mechanism Fig. An antibody titer follow-up study shows that PNp-induced antibodies specific to human M2e, PR8, and Aic were durable up to 4 months after the last immunization in mice Fig.
Mice were immunized with different PNps.
Double-layered protein nanoparticles induce broad protection against divergent influenza A viruses
Antibody endpoints against 4MtG, formalin-inactivated PR8 H1N1 and formalin-inactivated Aic H3N2 were measured in immune sera at time points leii 1 and 4 months after immunization. An influenza vaccine which induces broad cross protection against various influenza viruses has been a scientific challenge for more than a half century 40 A promising approach is to express a standalone HA stalk domain without the highly immunogenic and strain-specific head domain, and a stabilized M2e with high epitope density.
It was reported that HA stalk sera strongly bind HA from the same phylogenetic group but not the heterosubtypic HA from the different groups. Increasing evidence also showed that influenza genome of human isolates contained Lie gene segment from swine and avian origins, like p09 H1N1, human H5N1, and H7N9.
Therefore, we started generating HA stalk constructs from PR8 H1 group 1 and Aic Lie group 2as well as the M2e construct expressing tandem copies of M2e from human, swine, avian, and domestic fowl influenza.
Our study provides insights into a novel format of particulate influenza vaccines, which contain only antigenic proteins of interest and can induce robust and long-lasting immunity. We fabricated PNps approximately the size of the influenza A virions with a core of M2e displaying a shell of conserved hrHA domains. The binding of soluble hrHA to the desolvated PNps is speculated to be mediated through interaction of hydrophobic residues and fixed by DTSSP crosslinking primary amines.
To our knowledge, this design avoids the risk of instability shown by virus-like particles under osmotic stresses or during changes in salt concentration and prevents off-target immune responses against self-assembly motifs, such as the ferritin 1729 or hepatitis B core 35 protein used in some PNp designs.
DTSSP is a water-soluble, thiol-cleavable and primary amine-reactive cross-linker